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Full-Length 16S/18S/ITS Amplicon Sequencing

Introduction to Full-Length 16S/18S/ITS Amplicon Sequencing

Full-Length 16S/18S/ITS Amplicon Sequencing is a metagenomic approach that delves deep into the taxonomic composition of complex microbial communities. This sophisticated method amplifies full-length conserved genes, including 16S ribosomal RNA (rRNA), 18S rRNA, and Internal Transcribed Spacer (ITS) rRNA, using PCR techniques. The resulting amplicons are then subjected to high-fidelity, long-read sequencing on the PacBio Revio platform.

Full-length amplicon sequencing provides unparalleled taxonomic resolution. Unlike traditional methods that focus solely on hypervariable regions or intergenic regions, full-length amplicon sequencing captures the entire gene sequence. These amplicons range in length from 1-2kb, lengths that short-read sequencing cannot fully capture. This comprehensive approach reveals a richer, more detailed picture of microbial diversity, allowing for more accurate identification and classification of species within complex samples. Our 16S rRNA pipeline utilises PacBio’s Kinnex 16S rRNA library preparation kit to further improve accuracy and throughput.

PacBio Kinnex 16S rRNA Kit

The Kinnex kits are based on the MAS-Seq method, which concatenates smaller amplicons into larger fragment libraries for throughput increase. Novogene uses the Kinnex 16S rRNA kit as part of its 16S rRNA sequencing pipeline to further improve the throughput.

Applications of full-length amplicon sequencing

Amplicon-based metagenomic sequencing is an effective way to investigate microorganisms present in diverse samples. Its applications include:

  • Microbial diversity: characterise microbial communities in environmental samples and microbiome studies.
  • Taxonomic profiling: identify and classify microorganisms, reaching species-level.
  • Annotation of species as bacteria and archaea (16S), or fungi and other eukaryotes (ITS/18S).
  • Detection of pathogens and microbial contamination.
  • Analysis of samples from physiological fluids or tissues for microbiome profiling to assist in diagnosis and treatment.

Advantages of Novogene Full-Length 16S/18S/ITS Sequencing

Novogene has extensive experience with amplicon-based sequencing projects, and we are constantly innovating and adapting with the latest technology. Choose Novogene for your full-length amplicon sequencing project and benefit from the following advantages:

  • Targeted approach for the precise annotation and study of individual species within complex samples.
  • Our full-length amplicon sequencing service employs the MAS-seq library preparation method combined with PacBio’s HiFi sequencing technology, guaranteeing the delivery of high-quality data.
  • Our 16S rRNA pipeline utilises PacBio’s Kinnex 16S rRNA library preparation kit to further improve accuracy and throughput.
  • Our end-to-end service includes sample extraction, PCR amplification, library preparation, and Revio sequencing, along with ASV (DADA2) analysis for identification and functional annotation.

Microbial Metagenomics and Whole Genome Sequencing Service Flyer

In shotgun metagenomic sequencing, genomes from environmental samples are analysed, without the prior isolation and cultivation of individual species. Therefore, it is a powerful technique for studying microbial communities in their natural habitat. 16S/18S/ITS amplicon metagenomic sequencing is frequently used to identify and differentiate microbial species. Short (<500 bp) hypervariable regions of conserved genes are amplified by PCR and analysed using next generation sequencing (NGS) technology.

Download the Service Flyer

On-demand Webinar

Unfolding the Microbial World: A Journey through Sequencing Applications

The microbial world is teeming with an estimated one trillion species, only a fraction of which, approximately 155,000, have been sequenced so far. For researchers interested in the microbiome, the advent of next-generation sequencing (NGS) technologies and advanced data analysis methods presents an unparalleled opportunity to delve deeper into the intricacies of the microbial universe – whether you are interested in a single species or a microbial community. NGS serves as a pivotal tool, allowing us to dissect microbial communities, unravelling their complexities and unveiling their profound impact on diverse ecosystems.

In this webinar we will talk about myriad microbial sequencing options such as microbial whole genome sequencing, metagenomics, and metatranscriptomics. We will also discuss recent advances in the field such as the applications of long-read sequencing and highlight Novogene’s diverse microbial sequencing solutions to aid your research.

Learning Objectives:

  • Explore sequencing options for individual microbial species or communities
  • Discover recent advances in microbial sequencing technologies
  • Understand workflow and analysis pipelines for microbial sequencing.

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Long-read sequencing, the next era of genomics

In this webinar, guest speaker Thea Zieglar, Sequencing Application Sales Specialist at PacBio, and Shi-qi Ann, Deputy Product Marketing Director at Novogene, will guide you through HiFi long-read sequencing with the PacBio Revio.

Learning Objectives:

  • Understand the principle and advantages of highly accurate long-read sequencing to explore the full spectrum of genetic variation
  • Gain an overview of our most important applications, as well as possible options for whole genome sequencing, targeted sequencing, RNA sequencing and microbial genomics
  • Understand how PacBio long-read sequencing of extended 16S amplicons enables identification of metagenome community members at higher taxonomic resolution than common methods
  • Learn how Kinnex full-length RNA and single-cell RNA sequencing enables scientists to capture full-length isoforms, elucidating isoform diversity in development and disease.

Watch Now

Sample Requirements

Sample Type Amount
(Qubit)		 Concentration Volume Purity
Genomic DNA ≥ 200 ng ≥12ng/μL ≥20μL No degradation
No contamination/extra
fragments/color

Sequencing and Analysis

Sequencing Platform PacBio Revio
Read Length Full-length 16S/18S/ITS
Data Output 10K/20K/30K/50K clean reads
Standard Data Analysis
  • DADA2 denoise, ASV annotation and relative abundance analysis
  • Alpha diversity analysis (rarefaction curve, species boxplot)
  • Beta diversity analysis (heatmap, PCoA, PCA, NMDS)
  • Comparative analysis between groups and within group (Adonis, Anosim, t-test, Metastat, LDA)
  • Function prediction

Project Workflow of Novogene Amplicon Metagenomic Sequencing Services

The workflow for a new project begins with sample preparation and subsequent quality control. Once the quality of the DNA sample has been validated, PCR amplification will be performed on target regions (amplicons). The amplicons are then purified for library preparation, after which, library quality control commences. HiFi sequencing technology is used by the PacBio Revio platform to sequence the library and, once sequencing is complete and data quality control is performed, bioinformatics analysis is conducted to obtain high-quality results and create publication-ready figures.












Publications with Full-length Amplicon Metagenomic Sequencing

Full-length Amplicon Metagenomic Sequencing is a powerful tool for researchers aiming at the identification and differentiation of microbial species. By utilizing 16S/18S/ITS rRNA sequencing, this technique enables the characterization of microbial diversity in the environment through Alpha and Beta diversity analysis, facilitating the identification, description, and tracking of microorganisms of interest. Here is a list of featured publications that have employed Novogene’s Full-length Amplicon Metagenomic Sequencing services.

3D Principal Coordinate Analysis (PCoA) of Beta Diversity

3D Principal Coordinate Analysis (PCoA) of Beta Diversity

Principal Component Analysis (PCA) of relative
abundance

Principal Component Analysis (PCA) of relative

Non-Metric Multi-Dimensional Scaling (NMDS)

Non-Metric Multi-Dimensional Scaling (NMDS)

MetaStat Analysis between
Groups

MetaStat Analysis between

Cladogram of Linear Discriminant Analysis (LDA)

Cladogram of Linear Discriminant Analysis (LDA)

Histogram of LDA
Scores

Histogram of LDA

Beta Diversity Heatmap of Unweighted Unifrac Distance Matrix

Beta Diversity Heatmap of Unweighted Unifrac Distance Matrix

Heatmap of Taxonomic Relative
Abundance

Heatmap of Taxonomic Relative

Rarefaction Curves of Alpha Diversity

Rarefaction Curves of Alpha Diversity
Rarefaction Curves of Alpha Diversity

Anosim Analysis based on Unifrac
Distance

Anosim Analysis based on Unifrac

Histogram of Relative Abundance

Histogram of Relative Abundance

T-test Analysis of Species Differences between Groups

T-test Analysis of Species Differences between Groups

Species Accumulation Boxplot

Species Accumulation Boxplot

Venn Diagram of PICRUSt2 Results

Venn Diagram of PICRUSt2 Results

More Research Services

16S/18S/ITS Amplicon Metagenomic Sequencing

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